The overall goal of the proposed studies is to elucidate the role of the surface associated C-protein antigens in the pathogenesis of group B streptococcal (GBS) infections. GBS is the leading cause of neonatal sepsis and meningitis among infants in the United States; it is responsible for more than 50,000 cases per year of postpartum endometritis and for other serious invasive infections in immunocompromised adults. The development of a vaccine to prevent GBS infections has been hampered by a lack of understanding of the organism's virulence properties including the C proteins alpha and beta. The alpha C proteins display antiphagocytic properties, the beta C proteins bind specifically to human IgA, and both antigens contain protective epitopes and are potential candidates for a conjugate vaccine. The experimental plan is based on research completed and published under a FIRST Award and on preliminary studies providing important new data. The genes encoding alpha (bca) and beta (bcb) were cloned, expressed in Escherichia coli, and characterized by genetic, biochemical, and immunological techniques, including studies in animal models. A new protein named the epsilon antigen has recently been described and its gene cloned. A novel shuttle/suicide vector was developed to allow for more precise genetic analysis of virulence and immunity in GBS. Mutants in the bca gene are attenuated for virulence which suggests that the alpha antigen has an important role in the pathogenesis of GBS. These are apparently the first examples of site-specific genetic analysis of virulence in GBS. The specific aims of this proposal are (1) to determine the role of the C proteins alpha, beta, and epsilon in the pathogenesis of GBS infections by constructing selected mutations and testing virulence in vitro and in vivo models and (2) to define the function of structural and immunological variation among the C proteins, identify constant regions that may contain protective epitopes, and determine which regions elicit antibodies in human serum. Understanding of the range of and the molecular basis for the diversity of the C-protein antigens is essential for determining their role in the virulence of and immunity to GBS infections and in potential preventative strategies.